Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells exocytose cytotoxic granules to induce apoptosis of virally infected or tumor targets. Cytotoxic granules contain perforin, a pore-forming protein, and a group of serine proteases, called granzymes (Gzm), in a proteoglycan matrix. Perforin and either GzmA or GzmB is sufficient for granule-mediated lysis by CTL, but mice deficient in either Gzm may be impaired in defense against some viral infections. GzmB activates a ubiquitous apoptotic cascade induced by cleavage of caspases and can also directly cleave some key caspase pathway substrates. However, CTL granule mediated cytolysis is virtually unimpaired in the setting of complete caspase blockade. GzmA, the most abundant Gzm, activates a novel caspase-independent apoptotic pathway. The goal of this proposal is to continue to unravel the mechanism of action of GzmA, building on the research accomplished during the previous funding. GzmA, delivered into target cells via perforin, induces single-stranded DNA damage as Nell as apoptotic morphology and loss of cell membrane integrity. A special target of the GzmA cell death pathway is a 270-440 kDa endoplasmic reticulum-associated complex, called the SET complex, which contains three GzmA substrates, the nucleosome assembly protein SET, the DNA bending protein HMG-2 and the apurinic endonuclease (Ape1) in base excision repair (BER). GzmA cleavage of Ape1 makes target cell repair of GzmA-induced damage unlikely. The SET complex also contains the GzmA-activated DNase, NM23-H1, which is activated when GzmA cleaves its specific inhibitor, the SET protein. The competing renewal of this grant will investigate the kinetics of GzmA-mediated damage within target cells; determine the mitochondrial effects of GzmA; identify the remaining components of the SET complex and seek to understand further the role of the SET complex in normal cellular function and in GzmA-mediated cell death; and investigate the effect of GzmA on other DNA repair pathways, besides BER.